Project Summary: The hypothesis to be tested in this proposal is that exposure to heavy metals induces structural changes in alpha-synuclein protein, which leads to an accelerated formation of insoluble fibrillar aggregates. The studies proposed in this project are relevant for public health because they are focusing on the role of iron (Fe) and manganese (Mn) in the formation of a-synuclein fibrills, which plays a critical role in the onset and development of Parkinson disease. The specific aims of the proposed project are: Specific Aim 1: The proposed project will test the sub-hypothesis that electron microscopy and three-dimensional (3-D) reconstruction can generate a realistic model of the molecular arrangement into the alpha-synuclein fiber. The proposed research will (a) collect a high volume of high-resolution EM data of alpha-synuclein fibrils and (b) use a combination of single particle and helical reconstruction methods to combine all the information obtained from EM images into a 3-D structure of a-synuclein fibrils. Specific Aim 2: Negative stain EM, cryo-EM and three-dimensional reconstruction techniques will be used to elucidate the aggregation mechanism of alpha-synuclein in vitro (in the test tube). The proposed research will explore, by cryo-EM and three-dimensional reconstruction, the structure and morphology of the intermediate species formed during alpha-synuclein's self-assembly into fibers. Specific Aim 3: Negative stain EM, cryo-EM and three-dimensional reconstruction techniques will be used to test the sub-hypothesis that Fe and Mn exposure affects the aggregation pathway of alpha-synuclein in vitro (in the test tube). The proposed research will 1) further define the dose and time response relationship of Fe and Mn concentrations in alpha-synuclein fibrillation solution; 2) explore, by cryo-EM and three-dimensional reconstruction, the mechanistic actions of Fe and Mn on the fibrillation reaction of a-synuclein, and 3) use biochemical techniques to understand, in vitro, how heavy metal exposure influences the formation of alpha-synuclein aggregates. Relevance: There is substantial epidemiological evidence that occupational exposure to heavy metals can trigger the malfunction of alpha-synuclein. The research design outlined in this proposal has a high potential to make an important contribution to human health, both by improving our understanding of why the brain loses its function in a polluted environment and by suggesting new ways to treat these devastating illnesses.